tryopreservation Method
Zhe Lil*, Yanmei Ni3, Quannan Zhou!, Aihua Sun!, Xuemei Dai!, Tiandai Huang!, Huasun Huang!, Weifu Lint, Ludovic Lardee, Marc-Philippe Carron2, Pascal Montoro2
lRubber Research Institute, CATAS, Danzhou, 571737, Hainan, China
2UMR-1098 Biologie du développement des Espèces Pérennes, CIRAD, TA 80103, Avenue d'Agropolis, 34398 Montpellier Cedex 5, France
3College of Horticulture and Landscape Architecture of Hainan University, Danzhou, 571737, Hainan, China
*
E-mail: lizhecn@yahoo.cnABSTRACT
Friable and embryogenic calli could be induced from the inner integument of the rubber tree clone Reyan 88-13. And they had been proliferated and maintained for more than 2 years. Somatic embryos and further regeneration plantlets had been obtained from these calli. In order to preserve these calli for a long time and maintain their genetic characters, a liquid nitrogen vitrification cryopreservation method had been studied with these calli as the material. These calli were inoculated on the pre-culture medium, and had been cultured for 2-3 days in the dark, the temperature was 25-28 oC. Then the calli were immersed for 10-30 min in the 60% PVS2 solution at room temperature. After dehydration treatment, these calli were immersed for 20-40 min in PVS2 solution (28%-32% glycerol
+
12%-18% hexanediol+
12%-18% dimethyl sulphoxide+
136.9g1L sucrose) at O°C. Then the former PVS2 solution was removed, and new one was added. Afterward, these calli were thrown into liquid nitrogen (-196 OC) rapidly. The recovery of the calli was to take out of them from the liquid nitrogen quickly, immersed them in water for 2-3 min in which the temperature was 40 oC. After thaw and rinse, these calli were transferred on the subculture medium, and was cultured for 2-3 days in the dark, the temperature was 25-28 oC. Then they Were transferred on the fresh subculture medium and were cultured for 30 days. Most ofthe cryopreserved calli tumed brown and died. But sorne cell survived. So 30 days later, sorne tiny calli emerged, and grew. And they could be proliferated in the further subculture cycles. The post-thaw and survival calli could be used to induce somaticembryogenesis and further plant regeneration. The present results of the studies had the advantages that the experimental procedure was not complex, expensive apparatus could be avoided, repeatability and effect was good. So this method would be useful for long-term preservation of valuable germplasm materials, and would be beneficial for tissue culture and genetictransformation studies in Hevea brasiliensis.
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